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Abstract

Surface patterning is important in a wide spectrum of applications ranging from
microelectronics, sensors design and material science to high throughput screening, tissue
engineering and cell biology. A number of methods for specific patterning applications,
such as photolithography, soft lithography, or electron beam and dip-pen nanolithography,
have been developed. However, there is still a clear need for the development of novel
methods permitting patterning of different cell types, nano- and microparticles as well as
hydrogels incorporating cells. These novel patterning methods are vital for the
advancement of such research fields as tissue engineering, biomaterials and for
fundamental investigation of cell-cell communication, tissue and organ development.
The aims of this PhD thesis were: a) develop a technique for creating droplets of
liquid with defined geometries that can be used for patterning water soluble components;
b) optimize the conditions for the fabrication of porous polymer surfaces for the liquid
patterning; c) characterize the produced patterned polymer surfaces; d) further develop the
technique for maskless generation of liquid patterns with arbitrary geometry; e) optimize
the method for the patterning of different materials (chemicals, hydrogels, microparticles);
f) show an application of the method for patterning of living cells and characterize their
behavior on the composite surface during cultivation; g) show an application of the
technology to mimic natural cell-cell communication in vitro via signaling protein
propagation between patterned cell populations in co-culture.
The first part of the work was devoted to the development of porous polymer layers
with precise micropatterns of hydrophilic and hydrophobic areas. In order to fabricate
these patterns, UV-initiated photografting of 2,2,3,3,3-pentafluoropropyl methacrylate
(PFPMA) on porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)
(HEMA-EDMA) was optimized. Before and after photografting, both polymer substrates
were thoroughly characterized using water contact angle measurement, UV-Vis
spectroscopy, scanning electron microscopy (SEM) and time of flight secondary ion mass
spectrometry (ToF-SIMS). Porous properties were characterized by UV-Vis spectroscopy,
SEM and dynamic light scattering techniques (DLS). Due to the high difference in
wettability of the hydrophilic HEMA-EDMA polymer film and hydrophobic regions
coated with PFPMA polymer brushes, aqueous solutions can be trapped in the hydrophilic
areas, taking the shape of these areas. The transparency of the HEMA-EDMA monolith
originated from porous properties of the polymer makes it suitable for microscopic
monitoring of liquid patterns during experiments.
The method was for the first time applied for the simultaneous micropatterning of
multiple cell types. More than ten different cell populations separated by hydrophobic
borders could be cultured in microreservoirs. After adhesion, the cells could be placed in
the mutual culture medium, allowing cell-cell communication among populations. During
3 days co-culture in the mutual medium, cross-contamination was shown to be less than
1,5%, although the cells were pre-patterned in the hydrophilic areas separated by
hydrophobic borders of only two to three cell diameters. The capability of cell patterning
and long term cultivation opens the way for many interesting bio-applications, such as in vitro mimicking important biological processes that involve and depend on the
organization of multiple cell types into complex micropatterns in vivo. As a case study, I
together with Dr. Steffen Scholpp and Dipl. Eliana Stanganello (ITG, KIT) used the
developed technique to visualize spreading of signaling molecules (Wnt protein) from one
micropatterned population of fibroblast cells to another fibroblast population by activation
of the reporter system. Thus, we were able to simulate paracrine signaling system in vitro.
In addition, I further developed our technique into a new type of mask-less liquid
patterning or digital liquid patterning (DLP) method. The idea of this method is similar to
the working principle of a digital score board. A digital score board consists of many small
bulbs, which generate light symbols on it. In the case of DLP, instead of the bulbs, small
liquid droplets (digits) form a more complex liquid pattern on a substrate. The substrate for
DLP is a composite surface, consisting of a grid of hydrophilic HEMA-EDMA spots
divided by hydrophobic PFPMA barriers. The method allows on-demand fabrication of
liquid patterns without the need to change the substrate and use an additional photomask.
Patterns with customized geometries can be prepared manually by simply pipetting liquid
inside the spots and successively coalescing the generated droplets to form a liquid
micropattern. The DLP does not require clean room or high-precision microfabrication and
allows the manual positioning of microdroplets in the range of micrometer scale. It was
also shown that using superhydrophilic/superhydrophobic patterned surfaces leads to
spontaneous dewetting of the coalesced microdroplets on the interface of the
superhydrophobic border and the superhydrophilic spot. Hence, the usage of
hydrophilic/hydrophobic patterned surface ensures the stability of liquid patterns during
manipulations. Furthermore, the developed technique enables patterning of not only
solutions, e.g. different chemicals, but also suspensions of living cells and microparticles,
hydrogels, or formation of liquid multi-component gradients with complex geometries.
Thus, this method will be especially useful for biological studies, which require the
generation of complex patterns of different or the same cell types, or bioactive materials
and cellular gradients without the need for sophisticated microfluidic and printing
equipment, or for designing additional masks